- Penny Clouston, Consultant Clinical Scientist, Oxford University Hospitals NHS Foundation Trust
- Julie Evans, Registered Clinical Scientist, Oxford University Hospitals NHS Foundation Trust
Germline DNA is required for both cancer and rare disease referrals for the 100,000 Genomes Project. While most automated DNA extraction methods yield DNA from blood of sufficient quantity and quality for whole genome sequencing, there are particular specifications to adhere to.
Penny Clouston: In order to have the DNA accepted for the 100,000 Genomes Project, it has to be of exceptional high quality and also yield. And this is really important, because it means that we won’t be wasting valuable resources trying to sequence DNA that’s of a poor quality. It also means that we won’t have to re-bleed patients.
Penny Clouston: So the samples have to have an OD/260/280 ratio of 1.80 to 2.00. They have to have a minimum yield of DNA and it has to be high quality as well. It’s vital that we extract enough DNA, not only for the whole genome sequencing, but we also need to make sure we have an archive sample available in the laboratory. And this means that we can validate any findings from the whole genome projects on that sample.
Julie Evans: The blood sample for DNA extraction, it’s important not to freeze that before you do the DNA extraction because that can give you a lower yield. So the blood samples will be stored in the fridge until they’re extracted. For younger patients we might receive a smaller volume of blood, so it will be important to choose the extraction method best suited to the volume of blood that we receive.
Once you have extracted your DNA from the blood sample, according to your local procedures, it’s important to re-suspend the precipitated DNA in the correct buffer for an EDTA buffer pH 8 or the recommended proprietary buffer at the right volume to ensure that you achieve the correct final concentration of DNA. For whole genome sequencing, currently at least 10 micrograms at a concentration of 30 to 200 nanograms per microlitre of DNA must be shipped to the biorepository. Always check the latest specification.
In order to get reliable concentration and quality measurements, the final DNA solution must be homogeneous. This can be achieved by gentle agitation. Avoid vortexing the DNA or repeatedly pipetting up and down, as this may lead to unnecessary shearing, which could affect the quality of the sequencing results. For the 100,000 Genomes Project, Genomics England requires data on the concentration and purity of the extracted DNA to be sent with the samples to the biorepository.
Penny Clouston: The quality of the samples is really important. First of all they have to meet a very high standard so that they can actually be properly processed by the sequencing laboratory. If they don’t meet the standard, then they don’t go forward for that analysis.
So, what constitutes a high-quality sample for whole genome sequencing? There must be:
- DNA of high molecular weight with no evidence of shearing or degradation; and
- no significant protein contamination or carry-over of alcohol from the precipitation step.
Penny Clouston: Traditionally, UV spectrophotometry was used to measure the concentration and quality of DNA. However, this does have problems because it’s not only DNA that absorbs at OD 260, you can also get readings from other contaminants and proteins. So these days we tend to use a fluorescent dye which specifically binds to double-stranded DNA and then you can use fluorescence to detect this. So we tend to do the analysis on a machine called a Qubit using the picogreen dye and then you only get the reading from double-stranded DNA. So it’s much more accurate.
Having selected the appropriate assay type for the peripheral blood, or tumour sample, simply dilute the reagent mix with the buffer supplied in the kit according to the manufacturer’s instruction. Add your DNA sample and mix well. The picogreen dye binds rapidly and reaches equilibrium in less than 2 minutes. Fluorescence is detected by the fluorometer and is automatically converted to show DNA concentration by comparison with a standard curve.
To assess the purity of DNA and RNA, we measure the ratio of absorbance at wavelengths of 260 and 280 nanometres. A ratio of between 1.8 and 2.0 is the generally accepted value range. If the ratio is significantly lower, it may indicate the presence of protein, solvents or other contaminants that absorb strongly at or near 280 nanometres. In addition, it is recommended that you check that your extraction method produces DNA of high molecular weight. This can be undertaken on a representative sample from your recent extraction using agarose gel electrophoresis, a TapeStation or a bioanalyzer.
Here, you will see that DNA samples of varying quality have been run on 1% agarose gel for 2 hours. The profile for high-quality DNA is shown in lanes 1, 2 and 3. Whereas DNA that has been degraded is shown in lanes 4 and 5.